Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens.
Vera Gouvea,Roger I. Glass,P. A. Woods,Koki Taniguchi,H. F. Clark,Barbara Forrester,Zhao-Yin Fang +6 more
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TLDR
A PCR typing method was devised in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels, which provided a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.Abstract:
The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.read more
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Journal ArticleDOI
Molecular epidemiological survey of rotaviruses in sewage by reverse transcriptase seminested PCR and restriction fragment length polymorphism assay.
TL;DR: Both human and animal origins of rotavirus contamination of water seemed likely, as well as the presence of naturally occurring rotaviruses in raw sewage samples and treated effluent samples.
Journal ArticleDOI
Molecular and serologic characterization of novel serotype G8 human rotavirus strains detected in Blantyre, Malawi.
Nigel A. Cunliffe,Nigel A. Cunliffe,Nigel A. Cunliffe,Jon R. Gentsch,C. D. Kirkwood,Jailosi S. Gondwe,Winifred Dove,Osamu Nakagomi,Toyoko Nakagomi,Yasutaka Hoshino,Joseph S. Bresee,Roger I. Glass,Malcolm E. Molyneux,Malcolm E. Molyneux,C. Anthony Hart +14 more
TL;DR: The similarity of the VP7 gene sequences of the prototype strains described in this report to bovine serotype G8 rotaviruses suggests that they may represent human/bovine reassortant viruses.
Journal ArticleDOI
Rotavirus diarrhea in children and adults in a southern city of Brazil in 2003: distribution of G/P types and finding of a rare G12 strain.
Eduardo Pietruchinski,Fabrício José Benati,Flávio Lauretti,Jonas José Kisielius,Marli Ueda,Eduardo de Mello Volotão,Caroline C. Soares,Yasutaka Hoshino,Rosa Elisa Carvalho Linhares,Carlos Nozawa,Norma Santos +10 more
TL;DR: One rotavirus strain (HC91) bearing G12P[9] genotype with a “long” electropherotype was isolated from an 11‐month‐old boy with diarrhea for the first time in Brazil and was shown to belong to serotype G12 by neutralization.
Journal ArticleDOI
Direct detection of verotoxin-producing Escherichia coli in stool samples by PCR.
TL;DR: Testing of stool samples from patients with diarrhea showed a high concordance between PCR positivity and the presence of verotoxin-producing E. coli, determined by isolation of serotype O157:H7 on sorbitol-MacConkey medium.
Journal ArticleDOI
Secular variation in United States rotavirus disease rates and serotypes: implications for assessing the rotavirus vaccination program.
Daniel C. Payne,Peter G. Szilagyi,Mary A. Staat,Kathryn M. Edwards,Jon R. Gentsch,Geoffrey A. Weinberg,Caroline B. Hall,Aaron T. Curns,Haley Clayton,Marie R. Griffin,Gerry Fairbrother,Umesh D. Parashar +11 more
TL;DR: During the 2006 and 2007 rotavirus seasons, with only limited vaccine use, remarkable variability was observed in the population-based rates of severeRotavirus and in the rotav virus serotypes across the 3 sites.
References
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